1. Technical Field
The invention relates to a method for the examination of cells in a culture medium, in particular for in-situ microscopy in a bio-reactor, whereby cells in a sample volume, the depth of which is defined by windows in the direction of the optical axis of the microscope, are microscopically imaged and are automatically recorded and processed by means of an image processing system, and to an apparatus for implementing this method.
2. Prior Art
Such a method is disclosed by DE 40 23 002 C2, for example, and is particularly advantageous for use in bio-reactors which are also used for the cultivation of cells on an industrial scale. The automatic regulation of cell cultivation processes in the reactor requires measured values which provide information about the state of the culture medium. In principle, this method is not limited to organic cells but can also be used for the examination of other, inorganic particles in a medium, such as in an oil suspension or the like. For this reason, the term “cell” should be understood in its broadest sense, although reference will be made in the following to organic cells.
In the known method, the cells are imaged microscopically, with the image being captured by an automatic image processing system. The examined sample volume is defined in the direction of the optical axis of the microscope by windows, which ensure an unimpeded view of the sample volume.
Of special interest is the concentration, size and morphology of the cells. Furthermore, with in-situ microscopy it is possible to determine the cell size distribution, thus providing information for determining the anhydrous bio-mass. These parameters are supplied immediately by the described method, whereas offline analytic methods requiring the manual taking of samples are not suited for achieving a satisfactory regulation of the cell cultivation process.
However, it has proved difficult to obtain detailed information on the cell population in a bio-reactor because of the constraints imposed by the image processing system in recording all cells in the sample volume reliably. In order to examine as many cells as possible, the selected value for the distance between the windows and thus the depth of the sample volume must be as large as possible. This in turn is problematical, since the limited depth of field of the lens allows for only a narrow field of clear focus, and thus only a small number of cells can be imaged in clear detail and recorded by the image processing system in each measuring cycle. It is also difficult to differentiate individual cells when cells floating in suspension block each other from view. To insure that the image can be properly evaluated, it is necessary to use image processing software that is very expensive but does not always yield reliable results. In addition, some examination methods may require longer exposure times when imaging the cells. But this is not possible if the cells are able to move about freely in the sample volume.
On the other hand, if the sample volume is narrowed along the optical axis to a point where its thickness corresponds to the depth of field of the microscope lens, this results in very few cells being present within the sample volume for examination. If the sample volume is very narrow, this will also prevent an unimpeded flow of the culture medium between the windows.